Characterization of Rubus fruticosus mitochondria and salicylic acid inhibition of reactive oxygen species generation at Complex III/Q cycle: potential implications for hypersensitive response in plants

In addition to adenosine triphosphate (ATP) production, mitochondria have been implicated in the regulation of several physiological responses in plants, such as programmed cell death (PCD) activation. Salicylic acid (SA) and reactive oxygen species (ROS) are essential signaling molecules involved in such physiological responses; however, the mechanisms by which they act remain unknown. In non-photosynthesizing tissues, mitochondria appear to serve as the main source of ROS generation. Evidence suggests that SA and ROS could regulate plant PCD through a synergistic mechanism that involves mitochondria. Herein, we isolate and characterize the mitochondria from non-photosynthesizing cell suspension cultures of Rubus fruticosus. Furthermore, we assess the primary site of ROS generation and the effects of SA on isolated organelles. Mitochondrial Complex III was found to be the major source of ROS generation in this model. In addition, we discovered that SA inhibits the electron transport chain by inactivating the semiquinone radical during the Q cycle. Computational analyses confirmed the experimental data, and a mechanism for this action is proposed.

GABA accumulation and the hypersensitive response in isolated mesophyll cells treated with the G protein activator mastoparan

GABA (y-amino butyric acid) is a non-protein amino acid synthesized through the a-decarboxylation of L-glutamate. This reaction is catalyzed by L-glutamate decarboxylase (EC 4.1.1.15), a cytosolic Ca2+/calmodulin-stimulated enzyme. The purpose of this study is to determine whether or not GABA accumulation is associated with the hypersensitive response of isolated Asparagus sprengeri mesophyll cells. The addition of 25 J.lM mastoparan, a G protein activator, to suspensions of isolated asparagus mesophyll cells significantly increased GABA synthesis and cell death. Cell death was assessed using Evan's blue dye and fluorescein diacetate tests for cell viability. In addition, mastoparan stimulated pH-dependent alkalinization of the external medium, and a rapid and large 02 consumption followed by a loss of photosynthetic activity. The rate of 02 consumption and the net decrease in 02 in the dark was enhanced by light. The inactive mastoparan analogue Mas17 was ineffective in stimulating GABA accumulation, medium alkalinization, 02 uptake and cell death. Accumulation of H202 in response tomastoparan was not detected, however, mastoparan caused the cell-dependent degradation of added H202. The pH dependence of mastoparan-stimulated alkalinization suggests cellular electrolyte leakage, while the consumption of 02 corresponds to the oxidative burst in which 02 at the cell surface is reduced to form various active oxygen species. The results are indicative of the "hypersensitive response" of plants to pathogen attack, namely, the death of cells in the locality of pathogen invasion. The data are compatible with a model in which mastoparan triggers G protein activity, subsequent intracellular signal transduction pathway/s, and the hypersensitive response. It is postulated that the physiological elicitation of the hypersensitive response involves G protein signal transduction. The synthesis of GABA during the hypersensitive response has not been documented previously; however the role/s of GABA synthesis in the hypersensitive response, if any, remain unclear.

Gene-for-gene disease resistance without the hypersensitive response in Arabidopsis dnd1 mutant

The cell death response known as the hypersensitive response (HR) is a central feature of gene-for-gene plant disease resistance. A mutant line of Arabidopsis thaliana was identified in which effective gene-for-gene resistance occurs despite the virtual absence of HR cell death. Plants mutated at the DND1 locus are defective in HR cell death but retain characteristic responses to avirulent Pseudomonas syringae such as induction of pathogenesis-related gene expression and strong restriction of pathogen growth. Mutant dnd1 plants also exhibit enhanced resistance against a broad spectrum of virulent fungal, bacterial, and viral pathogens. The resistance against virulent pathogens in dnd1 plants is quantitatively less strong and is differentiable from the gene-for-gene resistance mediated by resistance genes RPS2 and RPM1. Levels of salicylic acid compounds and mRNAs for pathogenesis-related genes are elevated constitutively in dnd1 plants. This constitutive induction of systemic acquired resistance may substitute for HR cell death in potentiating the stronger gene-for-gene defense response. Although cell death may contribute to defense signal transduction in wild-type plants, the dnd1 mutant demonstrates that strong restriction of pathogen growth can occur in the absence of extensive HR cell death in the gene-for-gene resistance response of Arabidopsis against P. syringae.

The Arabidopsis thaliana RPM1 disease resistance gene product is a peripheral plasma membrane protein that is degraded coincident with the hypersensitive response

Disease resistance in plants is often controlled by a gene-for-gene mechanism in which avirulence (avr) gene products encoded by pathogens are specifically recognized, either directly or indirectly, by plant disease resistance (R) gene products. Members of the NBS-LRR class of R genes encode proteins containing a putative nucleotide binding site (NBS) and carboxyl-terminal leucine-rich repeats (LRRs). Generally, NBS-LRR proteins do not contain predicted transmembrane segments or signal peptides, suggesting they are soluble cytoplasmic proteins. RPM1 is an NBS-LRR protein from Arabidopsis thaliana that confers resistance to Pseudomonas syringae expressing either avrRpm1 or avrB. RPM1 protein was localized by using an epitope tag. In contrast to previous suggestions, RPM1 is a peripheral membrane protein that likely resides on the cytoplasmic face of the plasma membrane. Furthermore, RPM1 is degraded coincident with the onset of the hypersensitive response, suggesting a negative feedback loop controlling the extent of cell death and overall resistance response at the site of infection.

The Relationship between Photosynthesis and a Mastoparan-Induced Hypersensitive Response in Isolated Mesophyll Cells1

The G-protein activator mastoparan (MP) was found to elicit the hypersensitive response (HR) in isolated Asparagus sprengeri mesophyll cells at micromolar concentrations. The HR was characterized by cell death, extracellular alkalinization, and an oxidative burst, indicated by the reduction of molecular O2 to O2⋅−. To our knowledge, this study was the first to monitor photosynthesis during the HR. MP had rapid and dramatic effects on photosynthetic electron transport and excitation energy transfer as determined by variable chlorophyll a fluorescence measurements. A large increase in nonphotochemical quenching of chlorophyll a fluorescence accompanied the initial stages of the oxidative burst. The minimal level of fluorescence was also quenched, which suggests the origin of this nonphotochemical quenching to be a decrease in the antenna size of photosystem II. In contrast, photochemical quenching of fluorescence decreased dramatically during the latter stages of the oxidative burst, indicating a somewhat slower inhibition of photosystem II electron transport. The net consumption of O2 and the initial rate of O2 uptake, elicited by MP, were higher in the light than in the dark. These data indicate that light enhances the oxidative burst and suggest a complex relationship between photosynthesis and the HR.

Hypersensitive granulomatous response to mycobacterial glycolipid trehalose 6,6'-dimycolate

Protection against Mycobacterium tuberculosis infection requires an effective cell mediated immune response leading to granuloma formation and organism containment. Trehalose 6,6'-dimycolate (TDM), a glycolipid present on the mycobacterial cell wall, has been implicated as a key component in establishment of the granulomatous response. TDM has potent immunoregulatory and inflammatory properties; the acute response to TDM produces pathology resembling early Mycobacterium tuberculosis infection. We have further developed this model to study TDM-specific cell mediated immune responses that may play a role in the later stages of infection and pathology. Lungs from mice immunized with TDM in the form of a water-oil-water (w/o/w) emulsion demonstrate heightened histological damage, inflammation, lymphocytic infiltration, and vascular endothelial cell damage upon subsequent challenge with TDM. This exacerbated response can be adoptively transferred to naïve mice via transfer of non-adherent lymphocytes from TDM immunized mice. To identify the cell phenotype(s) regulating this response, purified non-adherent cell populations (CD4+ and CD8+ T cells; CD19 + B cells) were isolated from TDM immunized mice, adoptively transferred into naive mice, and subsequently challenged with TDM. Lung histopathology and cytokine production identified CD4+ cells as the critical cell phenotype regulating the TDM-specific hypersensitive response. The role of CD1d in presentation of TDM was examined. CD1d, a molecule known to present lipids to T cells, was identified as critical in development of the hypersensitive response. CD4+ cells were isolated from TDM-immunized CD1d -/- mice and adoptively transferred into naive wild type mice, followed by TDM challenge. These mice were deficient in development of the hypersensitive granulomatous response, signifying the importance of CD1d in the generation of TDM-specific CD4+ cells. The experiments presented in this dissertation provide further evidence for involvement of TDM-specific cell mediated immune response in elicitation of pathological damage during Mycobacterium tuberculosis infection. ^

Transcriptome Analysis of Capsicum Chlorosis Virus-Induced Hypersensitive Resistance Response in Bell Capsicum

Background Capsicum chlorosis virus (CaCV) is an emerging pathogen of capsicum, tomato and peanut crops in Australia and South-East Asia. Commercial capsicum cultivars with CaCV resistance are not yet available, but CaCV resistance identified in Capsicum chinense is being introgressed into commercial Bell capsicum. However, our knowledge of the molecular mechanisms leading to the resistance response to CaCV infection is limited. Therefore, transcriptome and expression profiling data provide an important resource to better understand CaCV resistance mechanisms. Methodology/Principal Findings We assembled capsicum transcriptomes and analysed gene expression using Illumina HiSeq platform combined with a tag-based digital gene expression system. Total RNA extracted from CaCV/mock inoculated CaCV resistant (R) and susceptible (S) capsicum at the time point when R line showed a strong hypersensitive response to CaCV infection was used in transcriptome assembly. Gene expression profiles of R and S capsicum in CaCV- and buffer-inoculated conditions were compared. None of the genes were differentially expressed (DE) between R and S cultivars when mock-inoculated, while 2484 genes were DE when inoculated with CaCV. Functional classification revealed that the most highly up-regulated DE genes in R capsicum included pathogenesis-related genes, cell death-associated genes, genes associated with hormone-mediated signalling pathways and genes encoding enzymes involved in synthesis of defense-related secondary metabolites. We selected 15 genes to confirm DE expression levels by real-time quantitative PCR. Conclusion/Significance DE transcript profiling data provided comprehensive gene expression information to gain an understanding of the underlying CaCV resistance mechanisms. Further, we identified candidate CaCV resistance genes in the CaCV-resistant C. annuum x C. chinense breeding line. This knowledge will be useful in future for fine mapping of the CaCV resistance locus and potential genetic engineering of resistance into CaCV-susceptible crops.

Natural variability in Arabidopsis thaliana germplasm response to Xanthomonas campestris pv. campestris

This work aimed to study the interaction between the model plant Arabidopsis thaliana and Xanthomonas campestris pv. campestris (Xcc), the pathogen responsible for black rot of crucifers. The response of 32 accessions of A. thaliana to the Brazilian isolate of Xcc CNPH 17 was evaluated. No immunity-like response was observed. "CS1308", "CS1566" and "CS1643" grown in continuous light were among the accessions that showed strongest resistance when inoculated with 5 x 10(6) CFU/mL. In contrast, "CS1194" and "CS1492" were among the most susceptible accessions. Similar results were obtained when plants were grown under short-day conditions. To quantify the differences in disease symptoms, total chlorophyll was extracted from contrasting accessions at different time points after inoculation. Chlorophyll levels from controls and Xcc inoculated plants showed a similar reduction in resistant accessions, whereas Xcc-inoculated susceptible accessions showed a greater reduction compared to controls. To test the specificity of resistance, accessions CS1308, CS1566, CS1643 and CS1438 (which showed partial resistance to CNPH 17), were inoculated with a more aggressive isolate of Xcc (CNPH 77) and Ralstonia solanacearum. Among the accessions tested, "CS1566" was the most resistant to Xcc CNPH 77 and also displayed resistance to R. solanacearum. Accessions CS1308, CS1566 and CS1643 were also inoculated with a high titer of Xcc CNPH 17 (5 x 10(8) CFU/mL). No collapse of tissue was observed up to 48 h after inoculation, indicating that a hypersensitive response is not involved in the resistance displayed by these accessions.

Molecular Characterization of the Oxalate Oxidase Involved in the Response of Barley to the Powdery Mildew Fungus1

Previously we reported that oxalate oxidase activity increases in extracts of barley (Hordeum vulgare) leaves in response to the powdery mildew fungus (Blumeria [syn. Erysiphe] graminis f.sp. hordei) and proposed this as a source of H2O2 during plant-pathogen interactions. In this paper we show that the N terminus of the major pathogen-response oxalate oxidase has a high degree of sequence identity to previously characterized germin-like oxalate oxidases. Two cDNAs were isolated, pHvOxOa, which represents this major enzyme, and pHvOxOb', representing a closely related enzyme. Our data suggest the presence of only two oxalate oxidase genes in the barley genome, i.e. a gene encoding HvOxOa, which possibly exists in several copies, and a single-copy gene encoding HvOxOb. The use of 3′ end gene-specific probes has allowed us to demonstrate that the HvOxOa transcript accumulates to 6 times the level of the HvOxOb transcript in response to the powdery mildew fungus. The transcripts were detected in both compatible and incompatible interactions with a similar accumulation pattern. The oxalate oxidase is found exclusively in the leaf mesophyll, where it is cell wall located. A model for a signal transduction pathway in which oxalate oxidase plays a central role is proposed for the regulation of the hypersensitive response.

Function of the oxidative burst in hypersensitive disease resistance.

Microbial elicitors or attempted infection with an avirulent pathogen strain causes the rapid production of reactive oxygen intermediates. Recent findings indicate that H2O2 from this oxidative burst plays a central role in the orchestration of the hypersensitive response: (i) as the substrate driving the cross-linking of cell wall structural proteins to slow microbial ingress prior to the deployment of transcription-dependent defenses and to trap pathogens in cells destined to undergo hypersensitive cell death, (ii) as a local threshold trigger of this programmed death in challenged cells, and (iii) as a diffusible signal for the induction in adjacent cells of genes encoding cellular protectants such as glutathione S-transferase and glutathione peroxidase. These findings provide the basis for an integrated model for the orchestration of the localized hypersensitive resistance response to attack by an avirulent pathogen.

Evidence for different QTL underlying the immune and hypersensitive responses of Eucalyptus globulus to the rust pathogen Puccinia psidii

The rust Puccinia psidii infects many species in the family Myrtaceae. Native to South America, the pathogen has recently entered Australia which has a rich Myrtaceous flora, including trees of the ecologically and economically important genus Eucalyptus. We studied the genetic basis of variation in rust resistance in Eucalyptus globulus, the main plantation eucalypt in Australia. Quantitative trait loci (QTL) analysis was undertaken using 218 genotypes of an outcross F2 mapping family, phenotyped by controlled inoculation of their open pollinated progeny with the strain of P. psidii found in Australia. QTL analyses were conducted using a binary classification of individuals with no symptoms (immune) versus those with disease symptoms, and in a separate analysis dividing plants with disease symptoms into those exhibiting the hypersensitive response versus those with more severe symptoms. Four QTL were identified, two influencing whether a plant exhibited symptoms (Ppr2 and Ppr3), and two influencing the presence or absence of a hypersensitive reaction (Ppr4 and Ppr5). These QTL mapped to four different linkage groups, none of which overlap with Ppr1, the major QTL previously identified for rust resistance in Eucalyptus grandis. Candidate genes within the QTL regions are presented and possible mechanisms discussed. Together with past findings, our results suggest that P. psidii resistance in eucalypts is quantitative in nature and influenced by the complex interaction of multiple loci of variable effect.

Structure-Function Analysis of the HrpB2-HrcU Interaction in the Xanthomonas citri Type III Secretion System

Bacterial type III secretion systems deliver protein virulence factors to host cells. Here we characterize the interaction between HrpB2, a small protein secreted by the Xanthomonas citri subsp. citri type III secretion system, and the cytosolic domain of the inner membrane protein HrcU, a paralog of the flagellar protein FlhB. We show that a recombinant fragment corresponding to the C-terminal cytosolic domain of HrcU produced in E. coli suffers cleavage within a conserved Asn264-Pro265-Thr266-His267 (NPTH) sequence. A recombinant HrcU cytosolic domain with N264A, P265A, T266A mutations at the cleavage site (HrcU(AAAH)) was not cleaved and interacted with HrpB2. Furthermore, a polypeptide corresponding to the sequence following the NPTH cleavage site also interacted with HrpB2 indicating that the site for interaction is located after the NPTH site. Non-polar deletion mutants of the hrcU and hrpB2 genes resulted in a total loss of pathogenicity in susceptible citrus plants and disease symptoms could be recovered by expression of HrpB2 and HrcU from extrachromossomal plasmids. Complementation of the Delta hrcU mutant with HrcU(AAAH) produced canker lesions similar to those observed when complemented with wild-type HrcU. HrpB2 secretion however, was significantly reduced in the Delta hrcU mutant complemented with HrcU(AAAH), suggesting that an intact and cleavable NPTH site in HrcU is necessary for total functionally of T3SS in X. citri subsp. citri. Complementation of the Delta hrpB2 X. citri subsp. citri strain with a series of hrpB2 gene mutants revealed that the highly conserved HrpB2 C-terminus is essential for T3SS-dependent development of citrus canker symptoms in planta.

Genetic transformation of Citrus sinensis cv. Hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker

Transgenic Citrus sinensis (L.) Osb. cv. Hamlin plants expressing the hrpN gene were obtained by Agrobacterium tumefaciens (Smith and Towns) Conn-mediated transformation. hrpN encodes a harpin protein, which elicits the hypersensitive response and systemic acquired resistance in plants. The gene construct consisted of gst1, a pathogen-inducible promoter, a signal peptide for protein secretion to the apoplast, the selection genes nptI1 or aacC1 and the Nos terminator. The function of gst1 in citrus was evaluated in transgenic C. sinensis cv. Valencia harboring the reporter gene uidA (gus) driven by this promoter. Histochemical analysis for gus revealed that gst1 is activated in citrus leaves by both wounding and inoculation with Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al. Genetic transformation was confirmed by Southern blot hybridization in eight cv. Hamlin acclimatized plants. RT-PCR confirmed hrpN gene expression in seven cv. Hamlin transgenic lines before pathogen inoculation. Some hrpN transgenic lines showed severe leaf curling and abnormal growth. Six hrpN transgenic lines were propagated and evaluated for susceptibility to X axonopodis pv. citri. RT-PCR confirmed gene expression in all six hrpN transgenic lines after pathogen inoculation. Several of the hrpN transgenic lines showed reduction in susceptibility to citrus canker as compared with non-transgenic plants. One hrpN transgenic line exhibited normal vegetative development and displayed very high resistance to the pathogen, estimated as up to 79% reduction in disease severity. This is the first report of genetic transformation of citrus using a pathogen-inducible promoter and the hrpN gene. Further evaluations of the transgenic plants under field conditions are planned. Nevertheless, the evidence to date suggests that the hrpN gene reduces the susceptibility of citrus plants to the canker disease. (C) 2009 Elsevier B.V. All rights reserved.

Oviposition by pierid butterflies triggers defense responses in Arabidopsis.

Insect eggs represent a threat for the plant as hatching larvae rapidly start with their feeding activity. Using a whole-genome microarray, we studied the expression profile of Arabidopsis (Arabidopsis thaliana) leaves after oviposition by two pierid butterflies. For Pieris brassicae, the deposition of egg batches changed the expression of hundreds of genes over a period of 3 d after oviposition. The transcript signature was similar to that observed during a hypersensitive response or in lesion-mimic mutants, including the induction of defense and stress-related genes and the repression of genes involved in growth and photosynthesis. Deposition of single eggs by Pieris rapae caused a similar although much weaker transcriptional response. Analysis of the jasmonic acid and salicylic acid mutants coi1-1 and sid2-1 indicated that the response to egg deposition is mostly independent of these signaling pathways. Histochemical analyses showed that egg deposition is causing a localized cell death, accompanied by the accumulation of callose, and the production of reactive oxygen species. In addition, activation of the pathogenesis-related1::beta-glucuronidase reporter gene correlated precisely with the site of egg deposition and was also triggered by crude egg extract. This study provides molecular evidence for the detection of egg deposition by Arabidopsis plants and suggests that oviposition causes a localized response with strong similarity to a hypersensitive response.

Fatty acid ketodienes and fatty acid ketotrienes: Michael addition acceptors that accumulate in wounded and diseased Arabidopsis leaves.

Physical damage and disease are known to lead to changes in the oxylipin signature of plants. We searched for oxylipins produced in response to both wounding and pathogenesis in Arabidopsis leaves. Linoleic acid 9- and 13-ketodienes (KODEs) were found to accumulate in wounded leaves as well as in leaves infected with the pathogen Pseudomonas syringae pv. tomato (Pst). Quantification of the compounds showed that they accumulated to higher levels during the hypersensitive response to Pst avrRpm1 than during infection with a Pst strain lacking an avirulence gene. KODEs are Michael addition acceptors, containing a chemically reactive alpha,beta-unsaturated carbonyl group. When infiltrated into leaves, KODEs were found to induce expression of the GST1 gene, but vital staining indicated that these compounds also damaged plant cells. Several molecules typical of lipid oxidation, including malonaldehyde, also contain the alpha,beta-unsaturated carbonyl reactivity feature, and, when delivered in a volatile form, powerfully induced the expression of GST1. The results draw attention to the potential physiological importance of naturally occurring Michael addition acceptors in plants. In particular, these compounds could act directly, or indirectly via cell damage, as powerful gene activators and might also contribute to host cell death.